MEDICAL
PROGRESS
NEWBORN SCREENING FOR METABOLIC DISORDERS
DEBORAH MARSDEN, MBBS, CECILIA LARSON, MD,
AND
HARVEY L. LEVY, MD
ur lives are often directed by chance occurrences. For Robert Guthrie, a lifelong interest in the cause of mental
retardation came from a retarded son and a dedication to preventing mental retardation in phenylketonuria (PKU)
came from the diagnosis of PKU in his wife’s mentally retarded niece.1,2 From these roots came Guthrie’s introduction
of newborn screening for PKU 3 and, subsequently, to the much more inclusive newborn screening for metabolic disorders of
today.
In this review, we endeavor to describe current newborn screening, the interrelationship between the public and private
sectors, the range of metabolic disorders that can be covered by screening, with emphasis on recent expansion using tandem mass
spectrometry (MS/MS), the reported outcomes of identified infants, and a number of issues that confront newborn screening.
O
HISTORICAL SETTING
Guthrie’s development of a bacterial inhibition assay for phenylalanine, and a newborn blood specimen dried on filter paper
to which the assay could be applied, made newborn screening possible.3 Of these two developments, however, it is the dried
blood specimen, the “Guthrie specimen,” that has been the more important in expanding newborn screening.4 Within a few years
after the Guthrie test for PKU initiated newborn screening,3 Guthrie had developed bacterial inhibition assays for other
metabolites so that additional metabolic disorders could be detected. These metabolites and disorders included leucine for maple
syrup urine disease, methionine for homocystinuria, and galactose for galactosemia.5 Beutler developed an enzyme assay for
galactosemia that was applied to the dried blood specimen,6 illustrating the central value in newborn screening of the unique
specimen as opposed to the bacterial assay. From then on, further expansions, such as screening for congenital hypothyroidism,7 sickle-cell disease,8 and congenital adrenal hyperplasia,9 have utilized methodologies other than the bacterial inhibition
assay.
The most recent of these applications is MS/MS. With the exception of a method of modified high-performance liquid
chromatography, which could detect PKU, tyrosinemia, and maple syrup urine disease disorders in a single analysis,10 all of the
methodologies introduced were for single disorders with very little if any coverage beyond that disorder. MS/MS is different in
that it includes a wide spectrum of metabolic disorders in a single assay.11 Notable among these are the disorders of organic acid
metabolism and fatty acid oxidation, which until recently were excluded from screening.
EXPANDED NEWBORN SCREENING
MS/MS has introduced a revolutionary advance in newborn screening for metabolic
disorders.12 MS/MS is a system of mass spectrometry in which two mass spectrometers
are placed in tandem, separated by a collision chamber (Figure 1; available at www.jpeds.
com). Blood eluted from a small disk of the newborn Guthrie spot is derivatized with
butanol and the butylated metabolites ionized by electrospray. The ions are separated by
charge within the first mass spectrometer and those selected by a computer program pass
into the central collision chamber where they are fragmented. The fragments then pass
into the second mass spectrometer where a scan identifies them as fragments from the
acylcarnitines of organic acids or fatty acids (mass of 85 Da) or fragments from amino
acids, identified by an additional scan as having lost a mass of 102 Da from the parent
ion.13 The concentration of each acylcarnitine and amino acid is determined by the ratio
FAOD
GA-I
GALT
IVA
MCADD
3-MCCD
Fatty acid oxidation disorders
Glutaric acidemia type I
Galactose-1-phosphate uridyltransferase
Isovaleric acidemia
Medium chain acyl-CoA dehydrogenase
deficiency
3-Methylcrotonyl-CoA carboxylase
deficiency
MS/MS
PKU
SCADD
VLCADD
Tandem mass spectrometry
Phenylketonuria
Short chain acyl-CoA dehydrogenase
deficiency
Very long chain acyl-CoA dehydrogenase
deficiency
From the Division of Genetics, Children’s
Hospital Boston; the Department of Pediatrics, Harvard Medical School, Boston; and
the New England Newborn Screening Program and the University of Massachusetts
Medical School, Worcester.
Supported by grants HG02085 from the
NIH/GRI and U22MC03959 from HRSA/
MCHB.
Submitted for publication Jul 22, 2005; last
revision received Nov 3, 2005; accepted
Dec 12, 2005.
Reprint requests: Dr Deborah Marsden,
Children’s Hospital Boston, 300 Longwood
Avenue, Fegan 10, Boston, MA 02115.
E-mail: [email protected].
edu.
J Pediatr 2006;148:577-84
0022-3476/$ - see front matter
Copyright © 2006 Elsevier Inc. All rights
reserved.
10.1016/j.jpeds.2005.12.021
577
PUBLIC HEALTH ROLE IN
NEWBORN SCREENING
The primary responsibility for newborn screening in the
United States, and in most other countries, resides in public
health, as emphasized by the Newborn Screening Task Force
in its 2000 report.14 From the inception of newborn screening, Guthrie recognized the need for public health to assume
this responsibility. He knew that only if public health agencies
conducted newborn screening would there be the organization and authority required to make the screening universal.
Hence, his initial collaboration was with Robert MacCready,
Director of the Diagnostic Division of the Massachusetts
Department of Public Health Laboratories. This resulted in
the beginning of population-based newborn screening.15
There are several reasons to consider newborn screening
a public health mission:
Figure 2. MS/MS profile of infant with medium chain acyl-CoA
dehydrogenase deficiency (MCADD) (bottom) compared with a normal
profile (top). The MCADD profile shows increased medium chain
acylcarnitines, including markedly increased C8 (octanoylcarnitine) and
increases in C6 (hexanoylcarnitine) and C10 (decanoylcarnitine), and
decreased levels of the long chain acylcarnitines C16
(hexadecanoylcarnitine) and C18 (octadecanoylcarnitine). (Reprinted with
permission from Chace DH, Kalas TA. Clin Biochem 2005;38:296-309.)
between the mass of the parent ion and the mass of a known
amount of corresponding stable isotope injected with each
specimen measured in the first mass spectrometer. The result
can be displayed as a print-out (Figure 2).
The list of disorders covered by MS/MS is extensive.
The precise number, however, depends upon definition. For
example, screening for increased phenylalanine is often considered as screening for one disorder, PKU, and is so considered in this review although at least four other disorders
characterized by hyperphenylalaninemia are also detectable.
Nevertheless, based on the reported results from several large
programs with several years experience of expanded screening,
at least 30 metabolic disorders are potentially identifiable by
MS/MS in the newborn (Table I; available at www.jpeds.
com). As noted, these cover a wide range of disorders that
include amino acids, carbohydrates, organic acids, and fatty
acid oxidation.
578
Marsden et al
1. Authority. Births occur in many hospitals, birthing centers,
and homes throughout a state. The only connection
among these individual entities is the state public health
agency, which has jurisdiction for licensing, regulating,
and inspecting birth facilities. Moreover, it is the role of
the state, through its public health agency, to enforce the
legal mandate for newborn screening that exists in all but
two US states.16
2. Organization. Only a single agency with authority can
organize the proper collection of blood specimens in the
many different locations and the timely transport of these
specimens to a testing laboratory.
3. Quality control. The metabolic disorders are individually
rare. Even each of the most frequent, PKU and medium
chain acyl-CoA dehydrogenase deficiency (MCADD) occurs in fewer than 1 in 10,000 infants.17,18 Thus, an
individual hospital laboratory, testing only the infants born
in that hospital, is unlikely to encounter an abnormal result
for most of these disorders during even a period of many
years. This lack of experience has been a significant cause
of missed cases.19 A central testing laboratory for a state or
region is likely to test large numbers of specimens and
thereby have the experience required to recognize abnormal results.
4. Efficiency. All newborn screening tests can be done by high
through-put methods. This high capacity sharply reduces
the unit costs of testing.
5. Follow-up. Following up of abnormal screening results
often requires outreach conducted by public health nurses
and maternal and child health agencies within state health
departments.
6. Responsibility. State public health departments have responsibility for determining which disorders should be
added to newborn screening. This responsibility and legal
authority is included in most of the state laws that mandate
screening,16 as illustrated by the process in Massachusetts
whereby MCADD was added to the list of mandated
disorders, and the rest of the expanded MS/MS panel was
added on a pilot basis. The process included the establishThe Journal of Pediatrics • May 2006
ment of an Advisory Committee and a public hearing
followed by a vote of the Advisory Committee and a final
decision by the Commissioner of Public Health.20,21
This is not to say that every newborn screening specimen must be tested only in a public health laboratory. Private
laboratories may serve to test specimens in a state or region in
collaboration with state public health agencies. In fact,
MS/MS was developed in a university laboratory 22 and was
pioneered by a private laboratory dedicated to newborn
screening that served most of the state of Pennsylvania and,
through contracts with public health agencies, also served
North Carolina and the District of Columbia.23 However, the
requirement for organized tracking of identified infants, clinical and laboratory confirmation, public education, and maintenance of confidentiality dictates the critical role of public
health in the success of the program.
SCREENING PROGRAMS
Coverage of metabolic and other disorders in the
United States is tracked by the National Newborn Screening
and Genetic Resource Center. The disorders included in each
state can be seen on their website.24 All states include screening for PKU, congenital hypothyroidism, and galactosemia,
and all but two states routinely screen for sickle-cell disease
and other hemoglobinopathies. Among the metabolic disorders, coverage for maple syrup urine disease, homocystinuria,
and biotinidase deficiency is included in approximately twothirds of the states. As of July 2005, newborn screening by
MS/MS was required or offered in 60% of the states to cover
many additional amino acid, organic acid, and fatty acid
oxidation disorders (FAOD). It is likely that within the near
future MS/MS will be employed by essentially all newborn
screening programs in the United States. A report prepared by
the American College of Medical Genetics, in collaboration
with the Health Resources and Services Administration of the
US Public Health Service and the American Academy of
Pediatrics, has recommended a panel of 29 disorders that
should be screened in all states.25 This report is currently
under consideration by the Secretary of Health and Human
Services. In most instances the testing is conducted in a state
public health laboratory. However, private laboratories in
some states, including Pennsylvania, Minnesota, Texas, and
California, perform this function under contract with the state
public health agencies. In addition, some states contract with
public health laboratories in other states to perform the testing function.24
Outside of the United States, universal newborn screening for metabolic disorders exists in almost all western European countries and in many of the eastern European countries. In Australasia, universal metabolic screening is
conducted in Japan, Australia, New Zealand, Singapore and
Taiwan; in the Middle East, Israel; and in South America,
Chile and Uruguay (Therrell BL, personal communication).
Newborn metabolic screening is especially comprehensive in
Japan, Australia, and New Zealand.
Newborn Screening For Metabolic Disorders
METABOLIC DISORDERS IN
NEWBORN SCREENING
Table I lists the screening abnormalities and metabolic
disorders that have been identified by newborn screening.
Featuring the abnormal analyte in the table is the most useful
method of presentation because this is the finding usually
reported as the abnormality to the physician. In addition to
the name of the possible disorder for each abnormal finding,
the table lists the name of the defective enzyme and a summary of the clinical features and treatment. This table can be
used as a quick reference when contacted by the newborn
screening program. More detailed and specific information
about follow-up of findings in expanded newborn screening
and the disorders can be found on the website of the New
England Consortium of Metabolic Programs.26
RESULTS OF EXPANDED
NEWBORN SCREENING
Frequency of Metabolic Disorders
Several large programs have conducted expanded newborn screening for a number of years, and reports of results
have begun to appear. As shown in Table II, the largest
experience has been that of Pediatrix Analytical (formerly
NeoGen Screening) located in Pennsylvania. In screening 1.1
million newborns, they found that 1 in 4000 newborns had a
confirmed metabolic disorder.18 The New England program
has reported essentially the same frequency of 1 in 4000
infants among 164,000 screened.27 In the Baden-Württemberg area of Germany, however, a higher frequency of 1 in
2400 among 250,000 screened neonates was found.17 In the
Australian state of New South Wales, the frequency of metabolic disorders among 362,000 screened newborns was 1 in
6000, but this frequency is substantially lowered by the exclusion of PKU from the data.28
The reported frequencies of disorders in three of the
four metabolic categories vary among the programs, but this
variation may be attributable in large part to the relatively
small number of infants screened in all but one of the programs and the rarity of many of the disorders. The frequency
of the fatty acid oxidation disorders, however, is remarkably
similar among the programs, with a range of 1 in 10,000 to 1
in 13,000. This is largely the result of a similar frequency
(1:16,000-1:21,000) reported for MCADD, which accounts
for the majority of cases (Table II).
Outcome of Identified Infants
Clinical follow-up of infants with metabolic disorders
identified by expanded screening has been reported from
Germany and New England. In the German study, Schulze et
al 17 found that 97 of the 106 infants (92%) identified in
Baden-Württemberg remained asymptomatic at a mean observation period of 13.5 months per child (range 0.1-38
months). Excluding the 36 infants who had a disorder that
they considered benign (eg, non-PKU hyperphenylalanine579
Table II. Frequencies of metabolic disorders and categories of disorders found by expanded newborn
screening in large programs
Frequencies
Program
No.
Screened
General
AA
UCD*
OA
FAOD
NeoGen/Pediatrix
Germany†
New England
Australia‡
1,100,000
250,000
164,000
362,000
1:4000
1:2400
1:4000
1:6000§
1:7500
1:4200
1:8200
-
1:230,000
1:42,000
1:82,000
1:60,000
1:28,000
1:15,000
1:55,000
1:30,000
1:13,000
1:10,000
1:10,000
1:12,000
AA, amino acid disorders; FAOD, fatty acid oxidation disorders; OA, organic acid disorders; UCD, urea cycle disorders.
*The most frequent UCD, ornithine transcarbamylase deficiency, and two other UCDs, carbamylphosphate synthetase and N-acetylglutamate synthetase deficiencies, are not usually
covered by expanded newborn screening.
†Baden-Württemberg area
‡State of New South Wales
§Excludes PKU
mia), they concluded that 61 infants or 1 in 4100 in their
screened population of 250,000 actually benefited from
screening and presymptomatic treatment.
In New England, Waisbren et al 29 compared outcome
in 50 children detected by expanded screening with 33 children diagnosed clinically who had the same disorders. The
children detected by screening began treatment a median of 4
months sooner than those clinically diagnosed and had a
significantly lower percentage of hospitalizations in the first 6
months of life (28% vs 55%; P ⫽ .02). In addition, the
medians of their developmental quotient were 14 points
higher on the mental index and 29 points higher in the motor
index than the clinically diagnosed group, and only two scored
in the mental retardation range, compared with 8 clinically
diagnosed children scoring in this range. The authors concluded that expanded newborn screening improved health
outcomes in children with metabolic disorders.
Despite these encouraging overall results, it is clear that
some children have not had good outcomes despite detection
by newborn screening. Two infants with MCADD in Pennsylvania died suddenly, one after an immunization and the
other during an intercurrent illness.30 In New England we
have also experienced two deaths in children with MCADD,
both associated with very mild intercurrent illness (Marsden
D, Shih VE, Waisbren SE, Levy HL, unpublished data).
Two of these four children were homozygous for the K329E
(often written as A985G) MCAD mutation, but the other
two were compound heterozygotes for this mutation and a
second rare mutation.
CHALLENGES AND ISSUES
Increased Detection by Screening
An important issue in expanded screening is the considerably larger number of detected cases of certain disorders
identified by screening as compared with the expected number based on clinical identification. Table III lists the magnitude of this difference among disorders in the reporting
programs. The greatest increase has been that of MCADD.
Wilcken et al 28 detected 17 infants with MCADD during 4
years of expanded screening as compared with 20 cases iden580
Marsden et al
Table III. Frequency of metabolic disorders in
expanded newborn screening compared with
clinical identification in New South Wales
and New England
Degree of increased frequency
Disorder
MCADD
VLCADD
SCADD
3-MCCD
GA-I
3-ketothiolase deficiency
Isovaleric acidemia
Citrullinemia
New South Wales* New England‡
5-fold
6-fold
31-fold
†
6-fold
19-fold
2-fold
†
4-fold
4-fold
2-fold
†
§
ND
†
†
*Annual frequency based on 4-year experience in expanded newborn screening compared with the previous 24 years of clinical identification.
†Only detected by screening; none identified clinically.
‡3.5 year experience in expanded newborn screening compared with clinical identification in New England states without expanded screening.
§Only detected clinically.
tified clinically in New South Wales during the previous 24
years before expanded screening. This represents a greater
than five-fold increase in the annual detection rate of
MCADD. They also reported large increases in the screening
detection rates of two other FAOD, very long chain acylCoA dehydrogenase deficiency (VLCADD), and short chain
acyl-CoA dehydrogenase deficiency (SCADD); and three
organic acid disorders, glutaric acidemia type I (GA-I),
3-methylcrotonyl-CoA carboxylase deficiency (3-MCCD)
and 3-ketothiolase deficiency. Waisbren and colleagues 29
reported a similar experience. Comparing the number of
infants detected by expanded screening in two New England
states with the number identified clinically in the other New
England states in which expanded screening had not yet
begun, they found a four-fold increase in MCADD detection
by expanded screening and two- to three-fold increases in the
screening detection rates of SCADD, VLCADD, 3-MCCD,
isovaleric acidemia (IVA), and citrullinemia.
The Journal of Pediatrics • May 2006
Although some of the excess detection by screening
could represent severe cases that were not diagnosed clinically
before newborn screening,31 it is likely that the greater part of
the excess is because of infants with benign or mild forms of
the disorders who might not come to clinical attention. For
instance, in a meta-analysis of reported studies of sudden
death because of MCADD in which the very frequent K329E
mutation was determined, the Centers for Disease Control
and Prevention found that the probability of sudden infant
death among those homozygous for this allele was 1% for the
United States and 3% for Europe and Australia but only 0.1%
among those with only one copy of the K329E allele.32 At
least 80% of cases of MCADD detected clinically, either
because of sudden death or episodes of hypoketotic hypoglycemia, have been homozygous for this allele.33 In newborn
screening, however, a lower percentage of the infants with
MCADD have been homozygous for this allele, ranging from
only 30% in Australia and in Massachusetts (Waisbren SE
and Levy HL, unpublished data) to 63% in Pennsylvania and
several other states.23 Accordingly, it is possible that many of
the infants detected by screening who have only one copy of
the K329E mutation have a mild or perhaps asymptomatic
form of MCADD.34 It should not be assumed, however, that
all children with only one K329E allele have mild disease, as
evidenced by two of the four children noted above who died
suddenly, by a child with severe disease identified in Australia,34 and by five infants who died suddenly and were diagnosed postmortem.35 Other metabolic disorders detected by
expanded newborn screening could represent a situation similar to that of MCADD. For instance, Spiekerkoetter et al 36
have identified a frequent mutation in asymptomatic patients
with VLCADD detected by expanded screening that may
indicate a mild form of the disorder, and Ensenauer et al 37
have identified a common mild, perhaps asymptomatic mutation also in patients with IVA detected by screening. Rhead
et al 38 have suggested from their follow-up in Wisconsin that
expanded newborn screening detects benign or very mild cases
of several disorders, including SCADD, 2-methylbutyrylCoA dehydrogenase deficiency, and 3-MCCD.
It is evident that the natural history of many of the
disorders detected by expanded screening is poorly understood. Because the clinical phenotype in some instances may
not be expressed until adulthood,39,40 only long-term follow-up will begin to provide this information.41,42
False-Positive Results (Transient “Abnormalities”)
False-positives will always occur in any population
screening program. They require repeat testing and are a
major problem in newborn screening, producing anxiety in
the families and additional work for physicians and screening
laboratories.11 The usual reason for a false-positive result is a
transient increase (or decrease) in the level of the measured
analyte, the frequency of which is a function of the cut-off
level for the analyte. This level is usually set arbitrarily, based
on the expected frequency of disorders in a given population
and the experience in testing unaffected newborns. The goal
Newborn Screening For Metabolic Disorders
is to identify all affected cases (sensitivity) without the burden
of excessive false-positives (specificity) that require repeat
testing and, in some cases, expensive confirmatory testing.
This is a delicate balance. Lowering the cut-off value may
produce greater sensitivity but also will result in reduced
specificity, therefore an increased number of false-positive
results. Most screening programs regularly review their cutoffs and revise them based on their experience. Other reasons
for false positive results include physiological variation in the
analyte level, enzyme immaturity in preterm infants, iatrogenic factors such as parenteral nutrition that may cause
elevation of several amino acids, and antibiotics that contain
a derivative of pivalic acid and produce pivaloylcarnitine,
which has the same MS/MS response as isovalerylcarnitine
(C5) and suggests isovaleric academia.43 Maternal factors may
produce elevations, including maternal PKU, which causes
transient neonatal hyperphenylalaninemia and 3-MCCD,
which causes a transient elevation of neonatal hydroxyisovalerylcarnitine (C5OH)44,45 Approximately 50% of falsepositive results are in neonatal intensive care unit babies.27
False-Negative Results
False-negative results or missed cases can be due to a
normal analyte level at the time the newborn specimen was
collected, such as can occur in screening for homocystinuria,46
or to the cut-off level. Two cases of GA-I were not detected
during the early introductory phases of MS/MS expanded
screening because the cut-off level had initially been set too
high28,47; these values were subsequently adjusted. In the
FAOD, especially the long chain defects, follow-up plasma
acylcarnitine confirmatory studies may be normal in an affected infant.48 However, measuring acylcarnitines in a dried
whole blood specimen instead of plasma for confirmation of
long chain defects may provide greater sensitivity and avoid
missing an infant. False-negative results may also be a result
of program or laboratory error.49 Because of the possibility of
a false-negative result, it should never be assumed that a
patient who presents with clinical disease cannot have a
metabolic disorder for which he presumably was screened as a
newborn.
Second-Tier Testing
A second test on the original screening specimen with
an abnormal primary screening result performed to specify
that the infant is affected before reporting by the laboratory is
known as second-tier testing. In galactosemia screening, for
instance, the screening specimen may be tested semi-quantitatively for galactose-1-phosphate uridyltransferase (GALT)
enzyme activity (the Beutler test6), when the primary screen
reveals increased galactose. The combination of increased
galactose and absence of GALT activity indicates classic
galactosemia. In screening for congenital hypothyroidism,
reduced thyroxine (T4) in the primary screen is followed by a
second-tier test for thyroid stimulating hormone that, if elevated, indicates congenital hypothyroidism. The versatility of
581
MS/MS makes it possible to utilize this technology for second-tier screening as well as for primary newborn screening.
An example is in screening for homocystinuria. An elevated
methionine level indicates the possibility of homocystinuria
because of a cystathionine ␤-synthase deficiency but can also
be because of methionine adenosyltransferase deficiency,
probably a benign disorder,50 or iatrogenically elevated because of parenteral nutrition. One program now measures
total homocysteine by MS/MS as a second-tier test to improve specificity.51 Another example is in screening for the
tyrosinemias in which a second-tier assay for succinylacetone
by MS/MS allows differentiation of tyrosinemia I from the
other causes of increased tyrosine in the neonate.52,53 Secondtier testing for markedly reduced or absent cortisol in screening for congenital adrenal hyperplasia separates the neonate
with congenital adrenal hyperplasia from the many more
infants who have only a transient elevation of 17-hydroxyprogesterone in the primary screen.54
Because MS/MS measures mass, compounds with the
same molecular weight cannot be separated. Examples are
leucine and isoleucine (both elevated in maple syrup urine
disease, a serious disorder) as well as hydroxyproline (elevated
in hydroxyprolinemia, a benign disorder). A second-tier test
using a different setting of the MS/MS can quantitate hydroxyproline on the basis of a fragment at m/z 68, which is
unique to that compound.55
Second-tier testing at the newborn screening level
markedly improves specificity. It also reduces the need for
repeat testing. In Minnesota where the Mayo group has
pioneered second-tier testing for MS/MS screening, the
false-positive rate is 0.08%56 as compared with false-positive
rates of 0.15% to 0.33% in other programs.17,27,28 In addition,
the predictive value of there being a disorder when a newborn
MS/MS screening result is reported as abnormal (positive
predictive value or PPV) in Minnesota is a very high 40%.56
Nevertheless, the additional costs of second-tier testing and
their most effective use require careful analysis.
DNA Screening
Mutation analysis can be performed on the newborn
filter paper specimen. This has sparked interest in primary
molecular screening.57 However, molecular testing has been
largely used for second-tier testing such as for cystic fibrosis,58
hemoglobinopathies,59 galactosemia,60 and GA-I.61 Despite
the interest in primary molecular screening, it is unlikely to
replace primary biochemical screening except in certain populations where there is a specific mutation in a disorder with
a high incidence. An example is in the Oji-Cree population of
western Canada where the incidence of GA-I is approximately 1 in 300 and a result of a single splice site mutation.62
Another example that has been proposed is for the “common”
mutations in glucose-6-phosphate dehydrogenase (G-6-PD)
deficiency in high-frequency areas.63 In some disorders, such
as MCADD, there is a very frequent mutation (K329E),
although many infants identified in newborn screening are
compound heterozygotes, often having a second mutation
582
Marsden et al
unique to that family. Many disorders have hundreds of
mutations, and new ones will continue to arise. Even with
microarray analysis for known mutations, secondary confirmatory testing will be still necessary.
Cost Effectiveness
The cost effectiveness of expanded newborn screening
is still being debated, but because even the longest programs
are only 6 to 7 years old, there are not yet any concrete data.
Reports utilizing meta-analysis and other modeling methods,
however, indicate that newborn screening is certainly costeffective for PKU and probably for MCADD and GA-I in
terms of reduced mortality and morbidity.64-68 The incremental cost of adding additional tests once the MS/MS
technology has been introduced is small. However, long-term
data will be necessary to determine the overall benefit. Nevertheless, as Grosse recently commented, public health is
about saving lives, preventing disability, and improving quality-of-life, not about financial savings.69
CONCLUSION
Undoubtedly, newborn screening has been one of the
most successful public health initiatives introduced in recent
times, with significant impact on mortality and morbidity in
countless children. To evaluate the long-term benefit of expanded newborn screening, however, it will be necessary to
ensure uniform screening in all states and develop collaborative protocols for diagnosis and follow-up. To meet those
needs, the American College of Medical Genetics has developed a uniform panel of recommended tests. This report has
been submitted for consideration by the Department of
Health and Human Services and is expected to be ratified
shortly. Protocols for evaluation of abnormal screens and case
definitions are being developed. New methodologies are currently being evaluated, and it is likely that in the future
newborn screening will be available for additional inborn
errors of metabolism, perhaps most imminently the lysosomal
storage disorders,70 but others as well.71
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35. Chace DH, DiPerna JC, Mitchell BL, Sgroi B, Hofman LF, Naylor
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36. Spiekerkoetter U, Sun B, Zytkovicz T, Wanders R, Strauss AW,
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37. Ensenauer R, Vockley J, Willard J-M, Huey JC, Sass JO, Edland SD,
et al. A common mutation is associated with a mild, potentially asymptomatic
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38. Rhead WJ, White A, Allain D, Lindh H, Hanson K, van Calcar S, et
al. Very-long-chain acyl-CoA dehydrogenase (VLCAD), 2-methylbutyryl-AD (2-MBAD), short chain-AD (SCAD), and 3-methylcrotonyl-CoA
carboxylase (3-MCC) deficiencies: newborn screening detects clinically benign or very mild cases. Molec Genet Metab 2004;81:153-86.
39. Losty H, Melville-Thomas G, Pollitt R, Davies S. Medium chain acyl
CoA deficiency sudden, unexpected death in a 23 year old woman. J Inherit
Metab Dis 2001;24(suppl 1):68.
40. Feillet F, Bollaert PE, deChillou C, Lefebvre E, Vianey-Saban C,
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and sever arythmia. J Inherit Metab Dis 2002;25(suppl 1):68.
41. Liebl B, Nennstiel-Ratzel U, Roscher A, von Kries R. Data required for
the evaluation of newborn screening programmes. Eur J Pediatr 2003;162:
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42. Dezateux C. Newborn screening for medium chain acyl-CoA dehydrogenase deficiency: evaluating the effects on outcome. Eur J Pediatr 2003;162:
S25-S28.
43. Abdenur JE, Chamoles NA, Guinle AE, Schenone AB, Fuertes ANJ.
Diagnosis of isovaleric acidaemia by tandem mass spectrometry: false positive
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Metab Dis 1998;21:646 –30.
44. Gibson KM, Bennett MJ, Naylor EW, Morton DH. 3-methylcrotonyl-coenzyme A carboxylase deficiency in Amish/Mennonite adults identified by detection of increased acylcarnitines in blood spots of their children.
J Pediatr 1998;132:519-23.
45. Koeberl DD, Millington DS, Smith WE, et al. Evaluation of 3-methylcrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry newborn screening. J Inherit Metab Dis 2003;26:25-35.
46. Wagstaff J, Korson M, Kraus JP, Levy HL. Severe folate deficiency and
pancytopenia in a nutritionally deprived infant with Homocystinuria caused
by cystathionine beta-synthase deficiency. J Pediatr 1991;118:569-72.
47. Smith WE, Millington DE, Koeberl DK, Lesser PS. Glutaric aciduria
Type I missed by newborn screening in an infant with dystonia following
promethazine administration. Pediatrics 2001;107:1184-7.
48. Fearing MF, Larson C, Strauss A, Marsden DL. Normal acylcarnitine
levels during confirmation of abnormal newborn screening in long-chain fatty
oxidation defects. J Inherit Metab Dis 2005;28:545-50.
49. Holtzman C, Slazyk WE, Cordero JF, Hannon WH. Descriptive
epidemiology of missed cases of phenylketonuria and congenital hypothyroidism. Pediatrics 1986;78:553-8.
50. Mudd SH, Levy HL, Tangerman A, Boujet C, Buist N, DavidsonMundt A, et al. Isolated persistent hypermethioninemia. Am J Hum Genet
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51. Cuthbert CD, Magera MJ, Hahn S, Tortorelli S, Rinaldo P, Matern D.
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583
mass spectrometry as a second tier newborn screening test for homocystinuria
and methylmalonic acidemias. J Inherit Metab Dis 2005;28(suppl. 1):8.
52. Magera MJ, Gunawardena ND, Hahn SH, Tortorelli S, Mitchell GA,
Goodman SI, Rinaldo P, Matern D. Quantitative determination of succinylacetone in dried blood spots for newborn screening of tyrosinemia type I.
Molec Genet Metab 2006, in press.
53. Allard P, Grenier A, Korson MS, Zytkovicz TH. Newborn screening
for hepatorenal tyrosinemia by tandem mass spectrometry: analysis of succinylacetone extracted from dried blood spots. Clin Biochem 2004;37:1010-15.
54. Lacey JM, Minutti CZ, Magena MJ, Tauscher AL, Casetta B, McCann M, et al. Improved specificity of newborn screening for congenital
adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry. Clin Chem 2004;50:621-5.
55. Chace DH, Hillman SL, Millington DS, Kahler SG, Roe CR, Naylor
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newborns by tandem mass spectrometry. Clin Chem 1995;41:62-8.
56. Rinaldo P, Tortorelli S, Hahn SH, Matern D. An objective score card
for reporting performance metrics of newborn screening by MS/MS (Abstract). Presented at 7th National Newborn Screening Meeting; October
26-29, 2005; Portland, Oregon.
57. Green NS, Pass KA. Neonatal screening by DNA microarray: spots
and chips. Nat Rev Genet 2005;6:147-51.
58. Comeau AM, Parad RB, Dorkin HL, Dovey M, Gerstle R, Haver K,
et al. Population-based newborn screening for genetic disorders when multiple DNA testing is incorporated: a cystic fibrosis newborn screening model
demonstrating increased sensitivity but more carrier detections. Pediatrics
2004;113:1573-81.
59. Bhardwaj U, Zhang Y-H, Jackson DS, Buchanan GR, Therrell BL,
McCabe LL, et al. DNA diagnosis confirms hemoglobin deletion in newborn
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60. Dobrowolski SF, Baras RA, Suzow JG, Berkley M, Naylor EW.
Analysis of common mutations in the galactose-1-phosphate uridyl transferase gene. J Mol Diagn 2003; 5:42-7.
61. Naylor EW, Chace DH, DiPerna JC, Kalas TA, Banas RA, Lin Z, et
al. Newborn and high risk screening for Glutaric aciduria type I: results of
584
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primary tandem mass spectrometry and 2nd-tier molecular testing in over 1.8
million specimens. Molec Genet Metab 2004;81:176.
62. Greenberg CR, Prasad AN, Dilling LA, Thompson JRG, Haworth JC,
Martin B, et al. Outcome of the first 3-years of a DNA-based neonatal
screening program for glutaric acidemia type I in Manitoba and northwestern
Ontario, Canada. Molec Genet Metab 2002;75:70-8.
63. Lin Z, Fontaine JM, Freer DE, Naylor EW. Alternative DNA-based
newborn screening for glucose-6-phosphate dehydrogenase deficiency. Molec
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64. Thomason MJ, Lord J, Bain MD, Chalmers RA, Littlejohns P, Addison GM, et al. A systematic review of evidence for the appropriateness of
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65. Venditti LN, Venditti CP, Berry GT, Kaplan PB, Kaye EM, Glick H,
et al. Newborn screening by tandem mass spectrometry for medium-chain
acyl-CoA dehydrogenase deficiency: a cost-effectiveness analysis. Pediatrics
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66. Pandor A, Eastham J, Beverely C, Chilcott J, Paisley S. Clinical
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newborn screening: pilot data and review. Pediatr Neurol 2002;26: 201-4.
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The Journal of Pediatrics • May 2006
Figure 1. Schematic representation of tandem mass spectrometry (MS/
MS). (Reprinted with permission from Fearing MK, Levy HL. Expanded
newborn screening using tandem mass spectrometry. Adv Pediatr 2003;50:
81-111.).
Newborn Screening For Metabolic Disorders
584.e1
Table I. Newborn screening overview
Screening analyte
Disorder
Enzyme defect
Neonatal/
Infantile features
(Potential)
General
treatment
Amino acid disorders
Phenylalanine
Phenylketonuria (PKU)
Phenylalanine hydroxylase
Leucine, valine*
Maple syrup urine disease
(MSUD)
Branched chain ␣ketoacid
dehydrogenase
complex
Hydroxyproline*
Methionine
Hydroxyprolinemia
Homocystinuria
Hydroxyproline oxidase
Cystathionine ␤-synthase
Methionine
Hypermethioninemia
(MAT I/III deficiency)
Methionine adenosyl
transferase (MAT I/III)
Tyrosine
Tyrosinemia type I
Fumarylacetoacetate
hydrolase
Tyrosine
Tyrosinemia type II
Tyrosine
aminotransferase
Tyrosine
Tyrosinemia III
Glycine
Nonketotic
hyperglycinemia (NKH)
4-hydroxyphenylpyruvate
dioxygenase
Glycine cleavage enzyme
Mental retardation
Autism
Hyperactivity
Seizures
Lethargy
Failure to thrive
Coma
Seizures
Maple syrup odor in
urine & cerumen
Benign
Mental retardation
Arachnodactyly
Osteoporosis
Ectopia lentis
Thromboembolism
Asymptomatic (?
rare cognitive
reduction)
Hepatic disease
Hypoglycemia
Hypophosphatemic
rickets
Keratoconjunctivitis
Palmar/plantar
keratosis
Cognitive reduction
Perhaps benign
Dietary restriction of
phenylalanine
Dietary restriction of
branched-chain
amino acids
None
Dietary restriction of
methionine;
supplemental B6
(for B6 responsive)
None known
NTBC (nitisinone);
dietary restriction
of tyrosine; liver
transplant
Dietary restriction of
phenylalanine &
tyrosine
Perhaps none
Seizures
Hypotonia
Marked lethargy
Supportive care;
sodium benzoate
Jaundice
Lethargy
Hepatomegaly
Coagulopathy
Aymptomatic (later
cataracts)
Asymptomatic or
identical to
galactosemia
Lactose-free diet
Carbohydrate Disorders
Galactose (or absence of GALT
activity)
Galactosemia
Galactose-1-phosphate
uridyltransferase
(GALT)
Galactose
Galactokinase deficiency
Galactokinase (GALK)
Galactose
Epimerase deficiency
Uridine diphosphate-4epimerase deficiency
(GALE)
Citrulline
Citrullinemia
Argininosuccinic acid
synthetase
Hyperammonemia
Mental retardation
Failure to thrive
Lethargy, coma
Citrulline
Citrin deficiency
Citrin (carrier)
Jaundice
Coagulopathy
Failure to thrive
Lactose-free diet
? None; ? lactosefree diet
Urea Cycle Disorders
584.e2
Marsden et al
Dietary restriction of
protein;
supplements: Larginine, sodium
benzoate, sodium
phenylacetate
Vitamin K; lipid
soluble vitamins
The Journal of Pediatrics • May 2006
Table I. Continued
Screening analyte
Disorder
Enzyme defect
Neonatal/
Infantile features
(Potential)
Citrulline
Argininosuccinic acidemia
Argininosuccinic acid
lyase
Hyperammonemia
Failure to thrive
lethargy, coma
Ataxia
Hepatomegaly
Arginine
Arginase deficiency
Arginase
Mild-moderate
hyperammonemia
Mental retardation
Spastic diplegia
C3 (propionylcarnitine)
Propionic acidemia
Propionyl-CoA
carboxylase
C3
Methylmalonic acidemia
Methylmalonyl-CoA
mutase
Metabolic acidosis
Hyperammonemia
Vomiting
Failure to thrive
Metabolic acidosis
Hyperammonemia
Vomiting
Failure to thrive
C3
cblC/D disorder
BLZ transport
C4 (butyrylcarnitine)
Isobutyryl-CoA
dehydrogenase
deficiency
Isovaleric acidemia
Isobutyryl dehydrogenase
Methylbutyryl-CoA
dehydrogenase
deficiency
Glutaric acidemia I
Methylbutyryl-CoA
dehydrogenase
C5OH (3-hydroxyisovalerylcarnitine)
3-Hydroxy-3methylglutaryl (HMG)CoA lyase deficiency
3-Hydroxy-3methylglutaryl-CoA
lyase
C5OH
3-Methylcrotonyl-CoA
carboxylase (3-MCC)
deficiency
3-Methylcrotonyl-CoA
carboxylase
C5OH
␤-ketothiolase deficiency
␤-ketothiolase
(mitochondrial
acetoacetyl-CoA
thiolase)
General
treatment
Dietary restriction of
protein;
supplements: Larginine, sodium
benzoate, sodium
phenylacetate
Dietary restriction of
arginine, protein
Organic Acid Disorders
C5 (isovalerylcarnitine)
C5
C5DC (glutarylcarnitine)
Newborn Screening For Metabolic Disorders
Isovaleryl-CoA
dehydrogenase
Glutaryl-CoA
dehydrogenase
Failure to thrive
Microcephaly
Clinical significance
uncertain
Metabolic acidosis
“Sweaty feet”
odor
Vomiting
Lethargy
Failure to thrive
Asymptomatic
Possible respiratory
distress
Dystonia
Macrocephaly
Metabolic acidosis
Vomiting
Lethargy
Hypotonia
Seizures
Hypoglycemia
Asymptomatic or
Lethargy
Hypotonia
Hypoglycemia
Lethargy
Hypoglycemia
Ketoacidosis
Dietary restriction of
threonine,
isoleucine, valine,
methionine
Dietary restriction of
isoleucine, valine,
methionine;
(supplemental B12
for B12-responsive
form)
Hydroxocobalamin
(Vitamin B12)
Carnitine
Dietary restriction of
leucine;
supplemental
glycine & Lcarnitine
Low-protein diet;
carnitine
Dietary restriction of
lysine &
tryptophan;
supplemental Lcarnitine, riboflavin
IV glucose; restrict
protein, fat
IV glucose; restrict
protein; often
supplemental
carnitine
Avoid fasting; lowprotein diet
584.e3
Table I. Continued
Screening analyte
C5OH
Disorder
Enzyme defect
Methylglutaconyl-CoA
hydratase deficiency
Holocarboxylase
synthetase deficiency
Methylglutaconyl-CoA
hydratase
Holocarboxylase
synthetase
Biotinidase deficiency
Biotinidase
C4 (butyrylcarnitine)
Short chain acyl-CoA
dehydrogenase
deficiency (SCADD)
Short chain acyl-CoA
dehydrogenase (SCAD)
C8 (octanoylcarnitine)
Medium chain acyl-CoA
dehydrogenase
deficiency (MCADD)
Medium chain acyl-CoA
dehydrogenase
(MCAD)
C8 Also C4, C5, C16, C18:1
Mutiple acyl-CoA
dehydrogenase
deficiency (MADD)
Also known as glutaric
acidemia II (GA-II)
C14 (tetradecanoylcarnitine)
C14:1 (tetradecenoylcarnitine)
Very long chain acyl-CoA
dehydrogenase
deficiency (VLCADD)
Multiple acyl-CoA
dehydrogenation
defects due to
decreased electron
transfer flavoprotein
(ETF), or decreased
electron transfer
flavoprotein ubiquinone
oxidoreductase
(ETF-Q0)
Very long chain acyl-CoA
dehydrogenase
(VLCAD)
C5OH
Biotinidase (reduced)
Neonatal/
Infantile features
(Potential)
General
treatment
Development delay
Low-protein diet
Vomiting
Tachypnea
Poor feeding
Developmental
delay, eczema,
seizures
Biotin
Biotin
Fatty Acid Oxidation Defects
584.e4
Marsden et al
Variable
presentation; may
be asymptomatic
With illness:
vomiting, lethargy,
metabolic
acidosis, seizures,
coma,
hypoglycemia,
metabolic acidosis
Asymptomatic when
well
With fasting and/or
illness: vomiting,
lethargy, seizures,
coma,
hypoketotic
hypoglycemia,
metabolic
acidosis,
hepatomegaly
Can cause sudden
death
Hypoketotic
hypoglycemia
Metabolic acidosis
“Sweaty feet” odor
Muscle weakness
Neonatal form has
occasional
dysmorphic
features &
hypotonia
Variable
presentation:
Cardiomyopathy
(hypertrophic)
Sudden death
With illness:
vomiting, lethargy,
seizures, coma
Hypoketotic
hypoglycemia
Avoid fasting Diet:
low-fat, highcarbohydrate
(CHO); often
supplemental
carnitine
Avoid fasting;
frequent CHO
feedings (q4 hours
until 4 months; q6
until 8 months; q8
thereafter); often
supplemental
carnitine
Avoid fasting; dietary
restriction of fat;
Frequent CHO
feedings;
supplement with
riboflavin; often
supplemental
carnitine
Avoid fasting; low-fat,
high-CHO diet;
frequent CHO
feedings; medium
chain triglycerides
(MCT); often
supplemental
carnitine
The Journal of Pediatrics • May 2006
Table I. Continued
Screening analyte
Disorder
Enzyme defect
C16 OH
(hydroxyhexadecanoylcarnitine)
C18:1 OH
(hydroxyoctadecenoylcarnitine)
C18 OH
(hydroxyloctadecanoylcarnitine)
Long chain hydroxyacylCoA dehydrogenase
deficiency (LCHADD)
Long chain hydroxyacylCoA dehydrogenase
(LCHAD)
C0 (carnitine) also C0/C16 ⫹
C18 ⫹ C18:1
Carnitine
palmitoyltransferase I
(CPT I) deficiency
Carnitine palmitoyl
transferase I (CPT I)
C0 (reduced)
Carnitine transporter
defect
Carnitine
palmitoyltransferase II
(CPT II) deficiency or
Carnitine/acylcarnitine
translocase deficiency
Carnitine transporter
C16 (palmitoylcarnitine)
C18:1 (octadecenoylcarnitine)
Carnitine palmitoyl
transferase II (CPT II) or
Carnitine/acylcarnitine
translocase
Neonatal/
Infantile features
(Potential)
Hypoketotic
hypoglycemia
Mild
hyperammonemia
Cardiomyopathy
Sudden death
Associated maternal
HELLP syndrome
Early severe
hypotonia
Renal tubular
acidosis
Lethargy
Fasting hypoketotic
hypoglycemia
Hepatomegaly
Seizures
Cardiomyopathy
Hypotonia
Infantile
presentation:
Hypoketotic
hypoglycemia
Lethargy
Seizures
Cardiomyopathy
General
treatment
Avoid fasting Diet:
low-fat, high-CHO;
supplemental with
MCT; often
supplemental
carnitine
Dietary restriction of
high-fat foods;
frequent CHO
feedings & MCT
Carnitine
Dietary restriction of
high-fat foods;
Frequent CHO
feedings; carnitine
supplement
*Leucine, isoleucine, and hydroxyproline have the same mass, so all produce the same MS/MS response. Defining the specific metabolite in this response requires additional testing.
Newborn Screening For Metabolic Disorders
584.e5